Applied and Environmental Microbiology最新文献

Microsporidia are unfriendly microorganisms, and their infections cause considerable damage to economically or environmentally important insects like silkworms and honeybees. Thus, the identification of measures to improve host resistance to microsporidia infections is critically needed. Here, an overexpressed miR-6498-5p transgenic silkworm line was constructed. Importantly, the survival rates and median lethal doses of the transgenic line were clearly higher after infection with Nosema bombycis. H&E staining and RT-qPCR analyses revealed an inhibitory effect on the proliferation of N. bombycis in the transgenic larvae. Metabolomics analysis further revealed the presence of 56 differential metabolites between the two lines. KEGG analysis of these 56 metabolites found that they were involved in various amino acid and vitamin metabolism pathways. Notably, VB6 metabolism was enriched among the metabolites, and the pathway was well known for its involvement in the synthesis, interconversion, and degradation of amino acids. These suggest that miR-6498-5p modifies parasitic environments to inhibit the proliferation of N. bombycis by affecting the host amino acid metabolism. These results demonstrate the potential of microRNAs as biomolecules that can promote resistance to microsporidia and provide new insights and a new approach to generate microsporidia-resistant biological materials.IMPORTANCEMicrosporidia have an extremely wide host range and are capable of infecting a wide variety of insects and vertebrates, including humans, and their lethality to multiple species often poses significant environmental management challenge. Here, we successfully constructed a microsporidium-resistant line in the silkworm, based on the overexpression of miR-6498-5p. Our results strongly support the hypothesis that miR-6498-5p efficiently suppresses the proliferation of Nosema bombycis by regulating the host VB6 metabolism, a key pathway for enzymes involved in amino acid transport and protein metabolism. Our study provides new insights for understanding host anti-pathogen defenses toward microsporidia.

Lactose assimilation is a relatively rare trait in yeasts, and Kluyveromyces yeast species have long served as model organisms for studying lactose metabolism. Meanwhile, the metabolic strategies of most other lactose-assimilating yeasts remain unknown. In this work, we have elucidated the genetic determinants of the superior lactose-growing yeast Candida intermedia. Through genomic and transcriptomic analyses, we identified three interdependent gene clusters responsible for the metabolism of lactose and its hydrolysis product galactose: the conserved LAC cluster (LAC12, LAC4) for lactose uptake and hydrolysis, the conserved GAL cluster (GAL1, GAL7, and GAL10) for galactose catabolism through the Leloir pathway, and a "GALLAC" cluster containing the transcriptional activator gene LAC9, second copies of GAL1 and GAL10, and a XYL1 gene encoding an aldose reductase involved in carbon overflow metabolism. Bioinformatic analysis suggests that the GALLAC cluster is unique to C. intermedia and has evolved through gene duplication and divergence, and deletion mutant phenotyping proved that the cluster is indispensable for C. intermedia's growth on lactose and galactose. We also show that the regulatory network in C. intermedia, governed by Lac9 and Gal1 from the GALLAC cluster, differs significantly from the galactose and lactose regulons in Saccharomyces cerevisiae, Kluyveromyces lactis, and Candida albicans. Moreover, although lactose and galactose metabolism are closely linked in C. intermedia, our results also point to important regulatory differences.IMPORTANCEThis study paves the way to a better understanding of lactose and galactose metabolism in the non-conventional yeast C. intermedia. Notably, the unique GALLAC cluster represents a new, interesting example of metabolic network rewiring and likely helps to explain how C. intermedia has evolved into an efficient lactose-assimilating yeast. With the Leloir pathway of budding yeasts acting like a model system for understanding the function, evolution, and regulation of eukaryotic metabolism, this work provides new evolutionary insights into yeast metabolic pathways and regulatory networks. In extension, the results will facilitate future development and use of C. intermedia as a cell-factory for conversion of lactose-rich whey into value-added products.

Agricultural crop yield losses and food destruction due to infections by phytopathogenic bacteria such as Burkholderia gladioli, which causes devastating diseases in onion, mushroom, corn, and rice crops, pose major threats to worldwide food security and cause enormous damage to the global economy. Biocontrol using bacteriophages has emerged as a promising strategy against a number of phytopathogenic species but has never been attempted against B. gladioli due to a lack of quantitative infection models and a scarcity of phages targeting this specific pathogen. In this study, we present a novel, procedurally straightforward, and highly generalizable fully quantitative ex planta maceration model and an accompanying quantitative metric, the ex planta maceration index (xPMI). In utilizing this model to test the ex planta virulence of a panel of 12 strains of B. gladioli in Allium cepa and Agaricus bisporus, we uncover substantial temperature-, host-, and strain-dependent diversity in the virulence of this fascinating pathogenic species. Crucially, we demonstrate that Burkholderia phages KS12 and AH2, respectively, prevent and reduce infection-associated onion tissue destruction, measured through significant (P < 0.0001) reductions in xPMI, by phytopathogenic strains of B. gladioli, thereby demonstrating the potential of agricultural phage biocontrol targeting this problematic microorganism.IMPORTANCEAgricultural crop destruction is increasing due to infections caused by bacteria such as Burkholderia gladioli, which causes plant tissue diseases in onion, mushroom, corn, and rice crops. These bacteria pose a major threat to worldwide food production, which, in turn, damages the global economy. One potential solution being investigated to prevent bacterial infections of plants is "biocontrol" using bacteriophages (or phages), which are bacterial viruses that readily infect and destroy bacterial cells. In this article, we demonstrate that Burkholderia phages KS12 and AH2 prevent or reduce infection-associated plant tissue destruction caused by strains of B. gladioli, thereby demonstrating the inherent potential of agricultural phage biocontrol.

Some strains of lactic acid bacteria can regulate the host's intestinal immune system. Bacterial cells and membrane vesicles (MVs) of Limosilactobacillus antri JCM 15950^T promote immunoglobulin A (IgA) production in murine Peyer's patch cells via toll-like receptor (TLR) 2. This study aimed to investigate the role of lipoteichoic acid (LTA), a ligand of TLR2, in the immunostimulatory activity of these bacterial cells and their MVs. LTA extracted from bacterial cells was purified through hydrophobic interaction chromatography and then divided into fractions LTA1 and LTA2 through anion-exchange chromatography. LTA1 induced greater interleukin (IL)-6 production from macrophage-like RAW264 cells than LTA2, and the induced IL-6 production was suppressed by TLR2 neutralization using an anti-TLR2 antibody. The LTAs in both fractions contained two hexose residues in the glycolipid anchor; however, LTA1 was particularly rich in triacyl LTA. The free hydroxy groups in the glycerol phosphate (GroP) repeating units were substituted by d-alanine (d-Ala) and α-glucose in LTA1, but only by α-glucose in LTA2. The dealanylation of LTA1 slightly suppressed IL-6 production in RAW264 cells, whereas deacylation almost completely suppressed IL-6 production. Furthermore, IL-6 production induced by dealanylated LTA1 was markedly higher than that induced by dealanylated LTA2. These results indicated that the critical moieties for the immunostimulatory activity of L. antri-derived LTA were the three fatty acid residues rather than the substitution with d-Ala in GroP. LTA was also detected in MVs, suggesting that the triacyl LTA, but not the diacyl LTA, translocated to the MVs and conferred immunostimulatory activity.

Importance: Some lactic acid bacteria activate the host intestinal immune system via toll-like receptor (TLR) 2. Lipoteichoic acid (LTA) is a TLR2 ligand; however, the moieties of LTA that determine its immunostimulatory activity remain unclear because of the wide diversity of LTA partial structures. We found that Limosilactobacillus antri JCM 15950^T has three types of LTAs (triacyl, diacyl, and monoacyl LTAs). Specifically, structural analysis of the LTAs revealed that triacyl LTA plays a crucial role in immunostimulation and that the fatty acid residues are essential for the activity. The three acyl residues are characteristic of LTAs from many lactic acid bacteria, and our findings can explain the immunostimulatory mechanisms widely exhibited by lactic acid bacteria. Furthermore, the immunostimulatory activity of membrane vesicles released by L. antri JCM 15950^T is due to the transferred LTA, demonstrating a novel mechanism of membrane vesicle-mediated immunostimulation.

The composition and stability of the microbial community structure of roots and root zone soils play a key role in the healthy growth of plants. We examined the distribution characteristics of phenolic acids and saponins, as well as microbial communities in the root space (root endosphere, rhizoplane soil, rhizosphere soil, and bulk soil) of healthy and root rot disease-affected Panax notoginseng. The results showed that after infection with root rot, the rhizoplane soil exhibited significant decreases in organic matter and hydrolyzable nitrogen and significant increases in available phosphorus, available potassium, and total nitrogen. The contents of phenolic acids (except benzoic acid) and ginsenoside Rg2 in the root endosphere significantly increased. Ferulic acid and p-hydroxybenzoic acid in the rhizoplane soil significantly increased. Rhodococcus increased significantly in the root endosphere, rhizoplane, and rhizosphere soil; Nitrospira decreased significantly in the rhizoplane, rhizosphere, and bulk soil; and Plectosphaerella decreased significantly in the root endosphere and rhizoplane soil. Moreover, the accumulation of most autotoxins can promote the growth of pathogens. In summary, the spatial autotoxic substances and microbial community differences in P. notoginseng roots jointly induce the occurrence of root rot.IMPORTANCEPanax notoginseng is highly susceptible to soil-borne diseases induced during planting, and root rot, which usually occurs in the root and stem parts of the plant, is the most severe. We divided the root environment of P. notoginseng into four parts (root endosphere, rhizoplane soil, rhizosphere soil, and bulk soil) and studied it with unplanted soil as the control. In this study, we examined the changes in the content of autotoxic substances in the root space of P. notoginseng, along with the interplay between these substances and microorganisms. This study revealed the mechanism underlying root rot and provided a theoretical basis for alleviating continuous cropping obstacles in P. notoginseng.

Grasslands are recognized as important reservoirs of soil biodiversity. Livestock grazing is implemented as a grassland management strategy to improve soil quality and enhance plant diversity. Soil microbial communities play a pivotal role in grassland ecosystems, so it is important to examine whether grazing practices affect the soil microbiome. Previous studies on grazing have primarily focused on bacteria and fungi, overlooking an important group-protists. Protists are vital in soil microbiomes as they drive nutrient availability and trophic interactions. Determining the impact of grazing on protists and their relationships with bacterial and fungal communities is important for understanding soil microbiome dynamics in grazed ecosystems. In this study, we investigated soil bacterial, fungal, and protist communities under four grazing levels: no grazing, moderate-use grazing, full-use grazing, and heavy-use grazing. Our results showed that heavy grazing led to a greater diversity of protists with specific groups, such as Discoba and Conosa, increasing in abundance. We also found strong associations between protist and bacterial/fungal members, indicating their intricate relationships within the soil microbiome. For example, the abundance of predatory protists increased under grazing while arbuscular mycorrhizal fungi decreased. Notably, arbuscular mycorrhizae were negatively associated with predatory groups. Furthermore, we observed that microbial network complexity increased with grazing intensity, with fungal members playing an important role in the network. Overall, our study reports the impact of temporal grazing intensity on soil microbial dynamics and highlights the importance of considering protist ecology when evaluating the effects of grazing on belowground communities in grassland ecosystems.

Importance: The significance of this study lies in its exploration of the effects of temporal grazing intensity on the dynamics of the soil microbiome, specifically focusing on the often-neglected role of protists. Our findings provide insights into the complex relationships between protists, bacteria, and fungi, emphasizing their impact on trophic interactions in the soil. Gaining a better understanding of these dynamics is essential for developing effective strategies for grassland management and conservation, underscoring the importance of incorporating protist ecology into microbiome studies in grasslands.

The symbiosis between Vibrio fischeri and the Hawaiian bobtail squid, Euprymna scolopes, is a tractable and well-studied model of bacteria-animal mutualism. Here, we developed a method to transiently colonize E. scolopes using D-alanine (D-ala) auxotrophy of the symbiont, controlling the persistence of viable infection by supplying or withholding D-ala. We generated alanine racemase (alr) mutants of V. fischeri that lack avenues for mutational suppression of auxotrophy or reversion to prototrophy. Surprisingly, an ∆alr mutant did not require D-ala to grow in a minimal medium, a phenomenon requiring metC, which encodes cystathionine β-lyase. Likewise, overexpression of metC suppressed D-ala auxotrophy in a rich medium. To block potential mechanisms of suppression, we combined the ∆alr mutation with deletions of metC and/or bsrF, which encodes a broad-spectrum racemase and investigated the suppression rates of four D-ala auxotrophic strains. We then focused on ∆alr ∆bsrF mutant MC13, which has a suppression rate of <10^-9. When D-ala was removed from a growing culture of MC13, cells rounded and lysed within 40 minutes. Transient colonization of E. scolopes was achieved by inoculating squid in seawater containing MC13 and D-ala, and then transferring the squid into water lacking D-ala, which resulted in loss of viable symbionts within hours. Interestingly, the symbionts within crypt 3 persisted longer than those of crypt 1, suggesting a difference in bacterial growth rate in distinct crypt environments. Our study highlights a new approach for inducing transient colonization and provides insight into the biogeography of the E. scolopes light organ.IMPORTANCEThe importance of this study is multi-faceted, providing a valuable methodological tool and insight into the biology of the symbiosis between Vibrio fischeri and Euprymna scolopes. First, the study sheds light on the critical role of D-ala for bacterial growth, and the underpinnings of D-ala synthesis. Our observations that metC obviates the need for D-ala supplementation of an alr mutant in minimal medium and that MetC-dependent growth correlates with D-ala in peptidoglycan, corroborate and extend previous findings in Escherichia coli regarding a role of MetC in D-ala production. Second, our isolation of robust D-ala auxotrophs led us to a novel method for studying the squid-Vibrio symbiosis, allowing for transient colonization without the use of antibiotics, and revealed intriguing differences in symbiont growth parameters in distinct light organ crypts. This work and the methodology developed will contribute to our understanding of the persistence and dynamics of V. fischeri within its host.

Bifidobacteria are recognized as health-promoting bacteria that reside in the human gut, helping in the digestion of fiber, preventing infections, and producing essential compounds like vitamins. To date, Bifidobacterium animalis subsp. lactis, together with Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, and Bifidobacterium longum, represents one of the species that are used as probiotic bacteria. Despite the extensive and detailed scientific research conducted on this microbial taxon, the molecular mechanisms by which B. animalis subsp. lactis exerts health benefits to its host are still largely unknown. Thus, we dissected the genetic repertoire and phylogenetic relationship of 162 strains of B. animalis subsp. lactis to select a representative reference strain of this taxon suitable for investigating its interaction with the host. The B. animalis subsp. lactis PRL2013 strain, which was isolated by a mucosal sample of a healthy adult, was chosen as the reference of the monophyletic cluster of human origin and revealed a greater adhesion index than that observed for another B. animalis subsp. lactis strain used in the industry as a probiotic supplement. Transcriptomics analyses of PRL2013 strain, when exposed to human cell monolayers, revealed 291 significantly upregulated genes, among which were found genes predicted to encode extracellular structures that may directly interact with human cells, such as extracellular polymeric substances, wall teichoic acids, and pili.

Importance: To date, many Bifidobacterium animalis subsp. lactis strains have been isolated from human fecal samples. However, their presence in these samples does not necessarily suggest an ability to colonize the human gut. Furthermore, probiotics of non-human origin may not effectively interact with the gut epithelium, resulting in transient bacteria of the gut microbiota. In vitro experiments with human cells revealed that B. animalis subsp. lactis PRL2013, an autochthonous member of the human gut, shows colonization capability, leading to future applications in functional foods.

Velvet proteins, as well as the epigenetic regulator LaeA, are conserved in numerous fungal species, where, in response to environmental cues, they control several crucial cellular processes, including sexual and asexual morphogenesis, secondary metabolism, response to oxidative stress, and virulence. During the last two decades, knowledge of their mechanism of action as well as understanding their functional roles, has greatly increased, particularly in Aspergillus species. Research efforts from multiple groups followed, leading to the characterization of other Velvet and LaeA homologs in species of other fungal genera, including important opportunistic plant and animal pathogens. This review focuses mainly on the current knowledge of the role of Velvet and LaeA function in fungal pathogenesis. Velvet proteins and LaeA are unique to fungi, and for this reason, additional knowledge of these critical regulatory proteins will be important in the development of targeted control strategies to decrease the detrimental impact of fungal pathogens capable of causing disease in plants and animals.

Contact with environmental microbial communities primes the human immune system. Factors determining the distribution of microorganisms, such as dispersal, are thus important for human health. Here, we used the relative number of bacteria shared between environmental and human samples as a measure of bacterial dispersal and studied these associations with living environment and lifestyles. We analyzed amplicon sequence variants (ASVs) of the V4 region of 16S rDNA gene from 347 samples of doormat dust as well as samples of saliva, skin swabs, and feces from 53 elderly people in urban and rural areas in Finland at three timepoints. We first enumerated the ASVs shared between doormat and one of the human sample types (i.e., saliva, skin swab, or feces) of each individual subject and calculated the shared ASVs as a proportion of all ASVs in the given sample type of that individual. We observed that the patterns for the proportions of shared ASVs differed among seasons and human sample type. In skin samples, there was a negative association between the proportion of shared ASVs and the coverage of built environment (a proxy for degree of urbanization), whereas in saliva data, this association was positive. We discuss these findings in the context of differing species pools in urban and rural environments.

Importance: Understanding how environmental microorganisms reach and interact with humans is a key question when aiming to increase human contacts with natural microbiota. Few methods are suitable for studying microbial dispersal at relatively large spatial scales. Thus, we tested an indirect method and studied patterns of bacterial taxa that are shared between humans and their living environment.